姬松茸子实体多糖及菌丝体液体培养的研究
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摘要
姬松茸，又名巴西蘑菇，是一种很有开发价值和应用前景的珍稀食药用真菌，它以其独特的营养价值和抗肿瘤、提高免疫力等药用功能而成为新近研究的热点。
本文研究了其子实体多糖的提取、纯化方法，对其理化性质作了初步鉴定。考虑到姬松茸菌丝体多糖也具有生理活性，食用菌菌丝体的液体培养更易实现工业化生产，本文对姬松茸菌丝体的液体培养条件也进行了探讨，具体研究工作和创新之处如下：
分别采用水法、草酸法、碱法和复合酶法对姬松茸子实体多糖进行提取，并比较了四种方法对多糖得率的影响及方法本身的优劣。结果表明，复合酶法为多糖提取的较优方法，其工艺参数为：酶解温度45℃，pH7.0，时间4h。
分别采用Sevag法、三氯乙酸法、三氯乙酸—正丁醇法、蛋白酶法对粗多糖中的蛋白质进行脱除，结果表明，三氯乙酸—正丁醇法和酶法效果较好。将两种方法相结合进行脱蛋白：在多糖溶液中添加适量蛋白酶，调pH至5.0，加入多糖含量2％的蛋白酶，40℃水浴酶解1h，再加入2倍于多糖体积的三氯乙酸—正丁醇液(v/v=1：10)，振摇20min，静置1.5h，重复三氯乙酸—正丁醇操作2次，此时蛋白去除率较高。
用活性炭、硅藻土、双氧水对多糖进行脱色处理时发现，用硅藻土脱色时多糖溶液颜色不变；活性炭有一定的脱色效果，但对多糖的吸附作用较强；双氧水的脱色效果较好，方法简便易行，且对多糖的吸附作用小，是一种理想的脱色方法。当加入多糖溶液体积2倍的双氧水时，脱色后多糖溶液的透光率达到95.5％。
通过复合酶法浸提、无水乙醇沉淀、酶法和三氯乙酸—正丁醇法结合去蛋白，双氧水脱色，从姬松茸子实体中得到粗多糖，再运用交联葡聚糖凝胶DEAE-Sephadex A-25，Sephadex G-200，对所得粗多糖进行纯化，得纯多糖(ABM)。
纯多糖ABM易溶于水、三氯乙酸等极性溶剂，极易溶于热水，难溶于非极性溶剂。经纸层析、柱层析、醋酸纤维薄膜电泳检测，表明ABM为均一性多糖。薄层层析检测得知ABM主要由葡萄糖和半乳糖组成，紫外和红外光谱表明，ABM中含有核酸物质，并含有少量结合蛋白。
对姬松茸液体培养条件研究，确定出菌丝生长的适宜碳源为蔗糖和小麦粉，氮源为牛肉膏和蛋白胨，pH范围为5—7，接种量为10％。因碳氮比对菌丝生长影响较大，采用二因子二次回归正交旋转组合设计研究了碳氮源对菌丝生长的综合影响，结果表明，液体基础培养基中添加3.64％的小麦粉，0.35％的牛肉膏时，菌丝生物量较大。采用正交试验对菌丝液体培养条件进行了优化，得到菌丝生长的优化工艺条件为：接种量为10％，pH为7，摇床转速为200r/min，温度为26℃。
本文的创新之处：
1．姬松茸脱蛋白、脱色方法未见报道，本文试验并比较了几种方法，确定了脱蛋白、脱色的较优方法和参数。
2.以往人们研究的多是姬松茸粗多糖的提取及其药理作用，本文对粗多糖进行
了纯化，并得到一种水溶性的均一多糖，且所得多糖与已报道的水溶性多糖在组成及
结构上有所不同。
3.对姬松茸生长的碳氮比进行了探讨，并综合优化了其菌丝体液体培养条件。
Agaricus blazei Murill, called Brazil Mushroom commonly, is a kind of edible and medicinal mushroom. It is getting research hotspot recently for its distinctive nutrient and medicinal values on tumor and immunity.
The polysacchande from Agaricus blazei Murill bodies was extracted and purified, and the physicochemical properties of which were determined. It was reported that the polysaccharides from Agaricus blazei Murill mycelia is effective against tumor. Moreover, the liquid-culture of mycelia will make production of polysaccharides industrialization more easily, so the liquid culture conditions of Agaricus blazei Murill mycelia were studied as well. The results and creative points are mainly as follow:
The polysaccharides from the fruiting bodies of Agaricus blazei Murill were extracted with hot water(100 ),oxalic acid (4 ),sodium hydroxide solution and compound enzyme respectively. Contrasting the effects of these methods on the extracting rate of the polysaccharides, the result showed compound enzyme method was a kind of better method and its optimum parameters were obtained. With the temperature of 45 ,pH of 70,and extract time of 3 hours ,the higher extracting rate of polysaccharides was reached.
Four schemes were used to remove protein from the extracted polysacchande and the results revealed that the scheme of enzyme and trichloroaceticacid -n-butanol were better than others. Combining these two schemes to remove protein, when the polysaccharides were enzymic hydrolysis for an hour in 40 癈 solution of pH 5.0, added trichloroaceticacid-n-butanol twice as much as polysacchande solution, stirred for 20 minutes, and put quietly for one and a half hour, the higher removal rate of protein was obtained.
Adopting active carbon, diatomite and hydrogen peroxide solution to decolourize the extracted polysacchande, it was found that the color of polysaccharide solution did not change with diatomite, the decolour effect of hydrogen peroxide solution was better than that of active carbon. The absorbance of polysaccharide by active carbon cause the polysaccharide content dropped. For hydrogen peroxide solution, there is no such phenomenon. So hydrogen peroxide solution is an ideal decolourant. When the hydrogen peroxide solution of two times as much as polysaccharide solution was added, the effect of decolour is the better, and the transmissivity of solution is 93.5%.
After treated with protein-removal and decoloure, the purified polysaccharide was obtained by further colume chromatography of DEAE-Sephadex A-25 and Sephadex G-200.
The purified polysaccharide (ABM) is prone to dissolving in polar solvent and difficult to dissolve in non-polar solvent ,and its homogeneity was verified by gel filtration, paper and
the electrophoresis of acetic acid fiber membrane . The components of ABM were identified by paper chromatography and UV and IR spectrum , and it was composed of D-Glc, D-Gal, nucleic acid, and a little protein.
The result of liquid culture of Agaricus blazei Murill showed that the optimum carbon source and nitrogen source were sucrose, wheat flour and beef creanK peptone respectively, the range of pH was from 5 to 7 and the spawn rate was 10%.
Considering the notable effects of ratio between carbon and nitrogen on the mycelia, response surface experiment were designed to analyze the comprehensive utilization of mycelia on carbon and nitrogen. The results revealed that when 3.64% wheat flour and 0.35% beef cream were added into the liquid culture medium, the biomass of mycelia reach the higher level.
By orthogonal experiments, the best technique conditions were obtained. The parameter were 10% of spawn rate, 7.0 of pH, 200r/min of rotation speed, and 26 'C of fermentation temperature.
The creative points are as following:
1 .The methods of removing protein and decolouration from Agaricus blazei Murill polysaccharides have not been reported .By contrasting several schemes, the better methods and parameters were determined in this paper.
2.The researches of Agaricus blazei Murill polysa
本文研究了其子实体多糖的提取、纯化方法，对其理化性质作了初步鉴定。考虑到姬松茸菌丝体多糖也具有生理活性，食用菌菌丝体的液体培养更易实现工业化生产，本文对姬松茸菌丝体的液体培养条件也进行了探讨，具体研究工作和创新之处如下：
分别采用水法、草酸法、碱法和复合酶法对姬松茸子实体多糖进行提取，并比较了四种方法对多糖得率的影响及方法本身的优劣。结果表明，复合酶法为多糖提取的较优方法，其工艺参数为：酶解温度45℃，pH7.0，时间4h。
分别采用Sevag法、三氯乙酸法、三氯乙酸—正丁醇法、蛋白酶法对粗多糖中的蛋白质进行脱除，结果表明，三氯乙酸—正丁醇法和酶法效果较好。将两种方法相结合进行脱蛋白：在多糖溶液中添加适量蛋白酶，调pH至5.0，加入多糖含量2％的蛋白酶，40℃水浴酶解1h，再加入2倍于多糖体积的三氯乙酸—正丁醇液(v/v=1：10)，振摇20min，静置1.5h，重复三氯乙酸—正丁醇操作2次，此时蛋白去除率较高。
用活性炭、硅藻土、双氧水对多糖进行脱色处理时发现，用硅藻土脱色时多糖溶液颜色不变；活性炭有一定的脱色效果，但对多糖的吸附作用较强；双氧水的脱色效果较好，方法简便易行，且对多糖的吸附作用小，是一种理想的脱色方法。当加入多糖溶液体积2倍的双氧水时，脱色后多糖溶液的透光率达到95.5％。
通过复合酶法浸提、无水乙醇沉淀、酶法和三氯乙酸—正丁醇法结合去蛋白，双氧水脱色，从姬松茸子实体中得到粗多糖，再运用交联葡聚糖凝胶DEAE-Sephadex A-25，Sephadex G-200，对所得粗多糖进行纯化，得纯多糖(ABM)。
纯多糖ABM易溶于水、三氯乙酸等极性溶剂，极易溶于热水，难溶于非极性溶剂。经纸层析、柱层析、醋酸纤维薄膜电泳检测，表明ABM为均一性多糖。薄层层析检测得知ABM主要由葡萄糖和半乳糖组成，紫外和红外光谱表明，ABM中含有核酸物质，并含有少量结合蛋白。
对姬松茸液体培养条件研究，确定出菌丝生长的适宜碳源为蔗糖和小麦粉，氮源为牛肉膏和蛋白胨，pH范围为5—7，接种量为10％。因碳氮比对菌丝生长影响较大，采用二因子二次回归正交旋转组合设计研究了碳氮源对菌丝生长的综合影响，结果表明，液体基础培养基中添加3.64％的小麦粉，0.35％的牛肉膏时，菌丝生物量较大。采用正交试验对菌丝液体培养条件进行了优化，得到菌丝生长的优化工艺条件为：接种量为10％，pH为7，摇床转速为200r/min，温度为26℃。
本文的创新之处：
1．姬松茸脱蛋白、脱色方法未见报道，本文试验并比较了几种方法，确定了脱蛋白、脱色的较优方法和参数。
2.以往人们研究的多是姬松茸粗多糖的提取及其药理作用，本文对粗多糖进行
了纯化，并得到一种水溶性的均一多糖，且所得多糖与已报道的水溶性多糖在组成及
结构上有所不同。
3.对姬松茸生长的碳氮比进行了探讨，并综合优化了其菌丝体液体培养条件。
Agaricus blazei Murill, called Brazil Mushroom commonly, is a kind of edible and medicinal mushroom. It is getting research hotspot recently for its distinctive nutrient and medicinal values on tumor and immunity.
The polysacchande from Agaricus blazei Murill bodies was extracted and purified, and the physicochemical properties of which were determined. It was reported that the polysaccharides from Agaricus blazei Murill mycelia is effective against tumor. Moreover, the liquid-culture of mycelia will make production of polysaccharides industrialization more easily, so the liquid culture conditions of Agaricus blazei Murill mycelia were studied as well. The results and creative points are mainly as follow:
The polysaccharides from the fruiting bodies of Agaricus blazei Murill were extracted with hot water(100 ),oxalic acid (4 ),sodium hydroxide solution and compound enzyme respectively. Contrasting the effects of these methods on the extracting rate of the polysaccharides, the result showed compound enzyme method was a kind of better method and its optimum parameters were obtained. With the temperature of 45 ,pH of 70,and extract time of 3 hours ,the higher extracting rate of polysaccharides was reached.
Four schemes were used to remove protein from the extracted polysacchande and the results revealed that the scheme of enzyme and trichloroaceticacid -n-butanol were better than others. Combining these two schemes to remove protein, when the polysaccharides were enzymic hydrolysis for an hour in 40 癈 solution of pH 5.0, added trichloroaceticacid-n-butanol twice as much as polysacchande solution, stirred for 20 minutes, and put quietly for one and a half hour, the higher removal rate of protein was obtained.
Adopting active carbon, diatomite and hydrogen peroxide solution to decolourize the extracted polysacchande, it was found that the color of polysaccharide solution did not change with diatomite, the decolour effect of hydrogen peroxide solution was better than that of active carbon. The absorbance of polysaccharide by active carbon cause the polysaccharide content dropped. For hydrogen peroxide solution, there is no such phenomenon. So hydrogen peroxide solution is an ideal decolourant. When the hydrogen peroxide solution of two times as much as polysaccharide solution was added, the effect of decolour is the better, and the transmissivity of solution is 93.5%.
After treated with protein-removal and decoloure, the purified polysaccharide was obtained by further colume chromatography of DEAE-Sephadex A-25 and Sephadex G-200.
The purified polysaccharide (ABM) is prone to dissolving in polar solvent and difficult to dissolve in non-polar solvent ,and its homogeneity was verified by gel filtration, paper and
the electrophoresis of acetic acid fiber membrane . The components of ABM were identified by paper chromatography and UV and IR spectrum , and it was composed of D-Glc, D-Gal, nucleic acid, and a little protein.
The result of liquid culture of Agaricus blazei Murill showed that the optimum carbon source and nitrogen source were sucrose, wheat flour and beef creanK peptone respectively, the range of pH was from 5 to 7 and the spawn rate was 10%.
Considering the notable effects of ratio between carbon and nitrogen on the mycelia, response surface experiment were designed to analyze the comprehensive utilization of mycelia on carbon and nitrogen. The results revealed that when 3.64% wheat flour and 0.35% beef cream were added into the liquid culture medium, the biomass of mycelia reach the higher level.
By orthogonal experiments, the best technique conditions were obtained. The parameter were 10% of spawn rate, 7.0 of pH, 200r/min of rotation speed, and 26 'C of fermentation temperature.
The creative points are as following:
1 .The methods of removing protein and decolouration from Agaricus blazei Murill polysaccharides have not been reported .By contrasting several schemes, the better methods and parameters were determined in this paper.
2.The researches of Agaricus blazei Murill polysa
引文
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